Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides
- Huang, JD
- Huen, MSY
- Cheah, KSE
- Lu, LY
- Tai, ACP
- Liu, DP
DOIAbstract
The λ phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner. Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as lon...
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